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Keywords:

  • α2-macroglobulin;
  • aspartic proteinase;
  • cathepsin D;
  • cathepsin E;
  • endolysosome system

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Purification of α2M from bovine serum
  6. Assays
  7. Sequencing of bovine α2M cDNA
  8. Gel electrophoresis and N-terminal sequence analysis
  9. Interaction of α2M with cathepsin E
  10. Analysis of synthetic peptides treated with cathepsin E and cathepsin D
  11. Results
  12. Effect of α2M on the hemoglobin hydrolyzing activity of cathepsin E and cathepsin D
  13. Effect of pH on the interaction of cathepsin E with α2M
  14. N-terminal sequencing of bovine α2M and sequencing its cDNA
  15. Analysis of the specific cleavage of α2M by cathepsin E with various synthetic peptides
  16. Structural changes in α2M upon complex formation with cathepsin E as determined by gel filtration analysis
  17. Discussion
  18. Acknowledgements
  19. References

α2-Macroglobulin (α2M) is an abundant glycoprotein with the intrinsic capacity for capturing diverse proteins for rapid delivery into cells. After internalization by the receptor- mediated endocytosis, α2M-protein complexes were rapidly degraded in the endolysosome system. Although this is an important pathway for clearance of both α2M and biological targets, little is known about the nature of α2M degradation in the endolysosome system. To investigate the possible involvement of intracellular aspartic proteinases in the disruption of structural and functional integrity of α2M in the endolysosome system, we examined the capacity of α2M for interacting with cathepsin E and cathepsin D under acidic conditions and the nature of its degradation. α2M was efficiently associated with cathepsin E under acidic conditions to form noncovalent complexes and rapidly degraded through the generation of three major proteins with apparent molecular masses of 90, 85 and 30 kDa. Parallel with this reaction, α2M resulted in the rapid loss of its antiproteolytic activity. Analysis of the N-terminal amino-acid sequences of these proteins revealed that α2M was selectively cleaved at the Phe811-Leu812 bond in about 100mer downstream of the bait region. In contrast, little change was observed for α2M treated by cathepsin D under the same conditions. Together, the synthetic SPAFLA peptide corresponding to the Ser808–Ala813 sequence of human α2M, which contains the cathepsin E-cleavage site, was selectively cleaved by cathepsin E, but not cathepsin D. These results suggest the possible involvement of cathepsin E in disruption of the structural and functional integrity of α2M in the endolysosome system.

Abbreviations
α2M

α2-macroglobulin

Hb

hemoglobin

LRP

low-density lipoprotein receptor-related protein

α2-Macroglobulin (α2M) is an abundant plasma glycoprotein composed of four identical subunits of Mr≈ 185 kDa [1]. α2M inhibits the activity of all classes of endopeptidases from both endogenous and foreign sources. The proteinases cleave an accessible region of the polypeptide chain of α2M, the bait region, thereby leading to the activation of internal thiol esters and the subsequent conformational change that entraps the responsible proteinase [1]. Then, the α2M-proteinase complexes are recognized by the low-density lipoprotein receptor-related protein (LRP)/CD91 on the surface of different cell types such as hepatocytes [2], fibroblast-like cells [3], and monocytes/macrophages [4] and become destined to rapid clearance and degradation in the endolysosome system [3,5]. Recent studies have also demonstrated that α2M has other important intrinsic capacity for capturing diverse molecules, including cytokines [6–9], growth factors [10–13], hormones [14], and soluble β-amyloid peptide [15], for rapid delivery into cells and degradation. The association of these molecules with α2M induces neither cleavage of the α2M peptide bond [16,17] nor the α2M conformational change [18,19]. The nature of this association is therefore distinct from the trapping mechanism for proteinases. However, these molecules bound to α2M are similarly targeted to cells expressing the α2M signaling receptor and become destined to rapid degradation in the endolysosome system. Therefore, α2M also plays an important part in the clearance of these molecules or regulates their biological activity. Meanwhile, α2M has also been shown to mediate immune responses through the delivery of foreign antigens to macrophages [20].

It is thus considered that α2M is involved in a wide range of physiological processes with the intrinsic capacity for capturing diverse target proteins for rapid delivery into cells and efficient degradation in the endolysosome system. However, the nature of the endocytosed α2M degradation in the endolysosome system is poorly understood. A physiological inactivator of α2M has not yet been identified. Cathepsins E and D are analogous endolysosomal aspartic proteinases in mammalian cells [21]. Cathepsin E represents a major portion of the proteolytic activity in the endosomal compartment in certain cell types such as macrophages and microglia [22–24], gastric cells [25], and antigen-presenting B cell lymphoblasts [26]. Cathepsin E is also associated with the plasma membrane of various cell types such as erythrocytes [27,28], osteoclasts [29], gastric parietal cells [30], renal proximal tubule cells [30], and hepatic cells [30]. On the other hand, cathepsin D is widely distributed in almost all the mammalian cells as the most abundant endolysosomal proteinase. More recently, it has been demonstrated that cathepsin E has a potential for foreign antigen processing for presentation by class II major histocompatibility complex [24] and possible regulation of activities of substance P and related tachykinins [31], whereas cathepsin D is indispensable for protection of the onset and development of a certain type of neuronal ceroid lipofucinosis [32] and proteolysis of proteins regulating cell growth and tissue homeostasis [33]. However, there is no unequivocal evidence for the participation of these two proteinases in the degradation of α2M. The inherent problem is that both cathepsins E and D were essentially inactive at around neutral pH where α2M is very stable, whereas they are most active at around pH 4.0 where α2M is unstable. Although previous work has suggested that cathepsin E is inhibited by α2M at pH 6.2 [34] and 5.5 [35], the ability of α2M to interact with cathepsins E and D below about pH 5.0 is still uncertain. In this report, we demonstrate that α2M is selectively associated with cathepsin E below pH 5.0 and rapidly cleaved it at a specific site distinct from the bait region, thereby losing its structural and functional integrity.

Materials

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Purification of α2M from bovine serum
  6. Assays
  7. Sequencing of bovine α2M cDNA
  8. Gel electrophoresis and N-terminal sequence analysis
  9. Interaction of α2M with cathepsin E
  10. Analysis of synthetic peptides treated with cathepsin E and cathepsin D
  11. Results
  12. Effect of α2M on the hemoglobin hydrolyzing activity of cathepsin E and cathepsin D
  13. Effect of pH on the interaction of cathepsin E with α2M
  14. N-terminal sequencing of bovine α2M and sequencing its cDNA
  15. Analysis of the specific cleavage of α2M by cathepsin E with various synthetic peptides
  16. Structural changes in α2M upon complex formation with cathepsin E as determined by gel filtration analysis
  17. Discussion
  18. Acknowledgements
  19. References

Trypsin (Type XIII) and human α2M were purchased from Sigma-Aldrich. Bovine liver cDNA was purchased from Clontech. Cathepsin E was purified from rat spleen [36] and human erythrocytes [27] as previously described. Cathepsin D was purified from rat [37] and bovine spleen [38] as described. The fluorogenic decapeptide substrate MOCAc-Gly-Lys-Pro-Ile-Ile-Phe-Phe-Arg-Leu-Lys(Dnp)-d-Arg-NH2 was synthesized as described previously [39]. Antibodies specific for rat cathepsin E and cathepsin D were raised in rabbits and purified by affinity chromatography as described previously [28]. Antiserum against bovine α2M was purchased from Yagai Research Center (Yamagata, Japan). All other chemicals were of reagent grade and were purchased from various commercial sources.

Purification of α2M from bovine serum

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Purification of α2M from bovine serum
  6. Assays
  7. Sequencing of bovine α2M cDNA
  8. Gel electrophoresis and N-terminal sequence analysis
  9. Interaction of α2M with cathepsin E
  10. Analysis of synthetic peptides treated with cathepsin E and cathepsin D
  11. Results
  12. Effect of α2M on the hemoglobin hydrolyzing activity of cathepsin E and cathepsin D
  13. Effect of pH on the interaction of cathepsin E with α2M
  14. N-terminal sequencing of bovine α2M and sequencing its cDNA
  15. Analysis of the specific cleavage of α2M by cathepsin E with various synthetic peptides
  16. Structural changes in α2M upon complex formation with cathepsin E as determined by gel filtration analysis
  17. Discussion
  18. Acknowledgements
  19. References

α2M was purified from bovine serum as described previously [40], with a slight modification. Briefly, the serum was applied to a Ni/nitrilotriacetic acid column (QIAGEN, 1.5 cm × 3.5 cm) and eluted with 50 mm sodium acetate buffer, pH 5.0, containing 50 mm NaCl. The eluate was applied to a Mono Q column equilibrated with 33 mm sodium phosphate buffer, pH 6.0. The column was washed with the same buffer and eluted with a linear gradient of NaCl (50–500 mm) in the buffer. Fractions containing α2M were determined by assaying the inhibitory activity against rat spleen extract at pH 3.8 using hemoglobin as a substrate. Fractions containing α2M, whose activity was determined by inhibition of the hemoglobin (Hb)-hydrolyzing activity of rat spleen extracts at pH 3.8, were pooled and concentrated and then subjected to gel filtration on Superose 6. The α2M fractions were pooled and subjected to the second Mono Q anion exchange chromatography. The pooled α2M fractions were concentrated and dialyzed against 20 mm Hepes buffer, pH 7.2, containing 140 mm NaCl.

Gel electrophoresis and N-terminal sequence analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Purification of α2M from bovine serum
  6. Assays
  7. Sequencing of bovine α2M cDNA
  8. Gel electrophoresis and N-terminal sequence analysis
  9. Interaction of α2M with cathepsin E
  10. Analysis of synthetic peptides treated with cathepsin E and cathepsin D
  11. Results
  12. Effect of α2M on the hemoglobin hydrolyzing activity of cathepsin E and cathepsin D
  13. Effect of pH on the interaction of cathepsin E with α2M
  14. N-terminal sequencing of bovine α2M and sequencing its cDNA
  15. Analysis of the specific cleavage of α2M by cathepsin E with various synthetic peptides
  16. Structural changes in α2M upon complex formation with cathepsin E as determined by gel filtration analysis
  17. Discussion
  18. Acknowledgements
  19. References

SDS/PAGE and immunoblotting were carried out following the procedure as described previously [28]. For the N-terminal amino-acid sequencing, the purified bovine α2M and the cathepsin E-digested protein were separated by SDS/PAGE (8% gel) under reducing conditions and then transferred onto poly(vinylidene difluoride) membranes and stained with Coomassie blue R-250. The stained bands were excised and the adsorbed proteins were subjected to an automatic protein/peptide sequencer (Applied Biosystems Model 477A).

Analysis of synthetic peptides treated with cathepsin E and cathepsin D

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Purification of α2M from bovine serum
  6. Assays
  7. Sequencing of bovine α2M cDNA
  8. Gel electrophoresis and N-terminal sequence analysis
  9. Interaction of α2M with cathepsin E
  10. Analysis of synthetic peptides treated with cathepsin E and cathepsin D
  11. Results
  12. Effect of α2M on the hemoglobin hydrolyzing activity of cathepsin E and cathepsin D
  13. Effect of pH on the interaction of cathepsin E with α2M
  14. N-terminal sequencing of bovine α2M and sequencing its cDNA
  15. Analysis of the specific cleavage of α2M by cathepsin E with various synthetic peptides
  16. Structural changes in α2M upon complex formation with cathepsin E as determined by gel filtration analysis
  17. Discussion
  18. Acknowledgements
  19. References

Synthetic peptides corresponding to the cleavage site region of α2M by cathepsin E were designed and custom-synthesized at the Peptide Institute (Osaka). Each peptide (20 nM) was incubated with or without either cathepsin E (20 pM) or cathepsin D (20 pM) in 0.1 m sodium acetate buffer, pH 3.8, and then subjected to reversed phase high-performance liquid chromatography (RP-HPLC) with a µBondapak C18 column (3.9 mm × 300 mm) (Waters). The column was eluted with a gradient of acetonitorile (0–60% in 30min) in 0.1% trifluoroacetic acid at a flow rate of 1.0 mL·min−1. Each peak fraction was pooled and the amino-acid sequence was analyzed by an Applied Biosystems automated derivatizer-analyzer (model 477A/120).

Effect of α2M on the hemoglobin hydrolyzing activity of cathepsin E and cathepsin D

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Purification of α2M from bovine serum
  6. Assays
  7. Sequencing of bovine α2M cDNA
  8. Gel electrophoresis and N-terminal sequence analysis
  9. Interaction of α2M with cathepsin E
  10. Analysis of synthetic peptides treated with cathepsin E and cathepsin D
  11. Results
  12. Effect of α2M on the hemoglobin hydrolyzing activity of cathepsin E and cathepsin D
  13. Effect of pH on the interaction of cathepsin E with α2M
  14. N-terminal sequencing of bovine α2M and sequencing its cDNA
  15. Analysis of the specific cleavage of α2M by cathepsin E with various synthetic peptides
  16. Structural changes in α2M upon complex formation with cathepsin E as determined by gel filtration analysis
  17. Discussion
  18. Acknowledgements
  19. References

To assess the inhibitory capacity of α2M from various sources for cathepsin D and cathepsin E, we first purified bovine α2M. The final preparation gave a single protein band with an apparent molecular mass of 170 kDa when analyzed by SDS/PAGE under reducing conditions (data not shown). The N-terminal amino-acid sequence of this protein was found to be AVDGKEPQYM, which was identical to the N-terminal sequence of the intact α2M as reported previously [41]. Addition of bovine α2M, as well as human α2M, to cathepsin E purified from human and rat sources (0.01 ng each) at pH 3.8 resulted in the significant decrease of Hb-hydrolyzing activity in a dose-dependent manner though the rate and extent of inhibition by bovine α2M were slightly but significantly higher than those by human α2M (Fig. 1). In contrast, little change was observed for the Hb-hydrolyzing activity of cathepsin D purified from rat and bovine spleen when α2M was added under the same conditions. This selective inhibition was further substantiated by experiments using the cell extract of rat spleen, in which cathepsin E and cathepsin D comprise 55 and 45% of the total Hb-hydrolyzing activity [25]. The cell extract treated with discriminative antibodies specific for cathepsin D to remove this protein showed a significant decrease in the Hb-hydrolyzing activity by addition of either human or bovine α2M, whereas the extract devoid of cathepsin E by immunoprecipitation with specific antibodies to cathepsin E showed no significant change in the Hb-hydrolyzing activity by each α2M (not shown). The results indicate that the selective reduction of cathepsin E activity by α2M occurs through the specific interaction between α2M and cathepsin E.

image

Figure 1. Effect of bovine α2M on the proteolytic activities of cathepsin E and cathepsin D. The Hb-hydrolyzing activity of cathepsin E purified from human erythrocytes and rat spleen and cathepsin D purified from bovine spleen and rat spleen (0.01 ng each) was measured at pH 3.8 in the absence or presence of increasing amounts of α2M.

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Effect of pH on the interaction of cathepsin E with α2M

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Purification of α2M from bovine serum
  6. Assays
  7. Sequencing of bovine α2M cDNA
  8. Gel electrophoresis and N-terminal sequence analysis
  9. Interaction of α2M with cathepsin E
  10. Analysis of synthetic peptides treated with cathepsin E and cathepsin D
  11. Results
  12. Effect of α2M on the hemoglobin hydrolyzing activity of cathepsin E and cathepsin D
  13. Effect of pH on the interaction of cathepsin E with α2M
  14. N-terminal sequencing of bovine α2M and sequencing its cDNA
  15. Analysis of the specific cleavage of α2M by cathepsin E with various synthetic peptides
  16. Structural changes in α2M upon complex formation with cathepsin E as determined by gel filtration analysis
  17. Discussion
  18. Acknowledgements
  19. References

Previous studies have shown that cathepsin E binds α2M at pH 6.2 and that the enzymatic activity of the complex toward the synthetic substrate Pro-Pro-Thr-Ile-Phe-Phe-(4-NO2)-Arg-Leu is not significantly affected [34]. Strong inhibition of the cathepsin E activity toward the protein substrate ribonuclease A by α2M was also observed at pH 5.5, where the α2M was cleaved by cathepsin E at the Phe-Tyr bond in the bait region [35]. Similarly, complete inhibition of the cathepsin D activity toward hemoglobin by α2M was observed at pH 6.2 [34]. These observations suggest that the inhibition of these enzymes by α2M at mild acidic pH values is similar to that observed with other classes of proteinases at neutral pH values. To assess whether the pH is crucial for the action of α2M on these aspartic proteinases, the effect of lowering the pH below 5.5 on the association of α2M with cathepsins E and D and the structural change in α2M upon complex formation with these enzymes were analyzed. After incubation of α2M at 37 °C for 1 h with or without cathepsin E at the indicated pH values, the reaction mixtures were analyzed by nondenaturing PAGE (Fig. 2). α2M treated with cathepsin E at pH 5.5 migrated faster than the native α2M, indicating that α2M became bound to cathepsin E and thereby underwent conformational change into the more compact form. α2M treated with cathepsin E at pH 4.5 was diffusely stained and its mobility was faster than that observed at pH 5.5. α2M treated with cathepsin E at pH 3.8 was seen near the dye front.

image

Figure 2. Association of cathepsin E with α2M under acidic conditions. Cathepsin E was incubated with bovine α2M at a molar ratio of 2 : 1 at 37 °C for 60 min at the indicated pH values. Then the reaction mixture was subjected to native PAGE at pH 8.9. As a control, α2M treated with trypsin (an α2M/enzyme ratio, 1 : 1.5) at pH 7.5 and 37 °C for 10 min was run on the same gel.

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To assess the mechanism of action of cathepsin E on α2M, we next analyzed whether the mobility changes of cathepsin E-treated α2M was due to cleavage of the bait region or degradation of bovine α2M. The cathepsin E-treated α2M molecule was rapidly degraded to generate two major protein bands with apparent molecular masses of 90 and 30 kDa within a 15-min incubation at pH 3.8 and 37 °C (Fig. 3A). The N-terminal amino-acid sequences of the 90- and 30-kDa peptides were found to be AVDGKEP and LAIPVE, respectively. Within a 30-min incubation, an additional 85-kDa peptide was generated and its N-terminal amino-acid sequence was identical to that of the 90-kDa peptide. All these peptides were further degraded by prolonged incubation. Similar results were obtained with human α2M (Fig. 3B), although the N-terminal amino-acid sequence of human 85-kDa peptide was not identical to that of the 90-kDa peptide from bovine α2M. The N-terminal amino-acid sequence of the 85-kDa peptide derived from human α2M corresponded to the sequence of the N-terminus of the original α2M, and the N-terminal amino-acid sequence of the 90-kDa was identical to the sequence starting with 685th Tyr (YESDVM). Although human α2M treated with cathepsin E generated the 30-kDa peptide, its N-terminal amino-acid sequence could not be determined by overlapping of additional peptides in the vicinity of 30 kDa. These three peptides were also generated from cathepsin E-treated human α2M at pH 4.5 and 5.5 (Fig. 3C). As no detectable accumulation of the other protein bands was observed, cleavage at the other sites probably much more rapid than at the one that was slow enough to allow the cleaved fragments to build up. This may explain some discrepancy in molecular sizes between the intact 170-kDa polypeptide and the generated 90- and 30-kDa fragments. In contrast, no degraded protein bands were observed for the cathepsin D-treated bovine α2M under the same conditions (Fig. 3A). Figure 4 shows SDS/PAGE profiles of bovine α2M treated with cathepsin E at different molar ratios at 37 °C and 30 min at pH 3.8. The α2M-cathepsin E complex gave the 90- and 30-kDa peptides at a α2M/cathepsin E molar ratio of below 10. More than 90% of the original α2M disappeared at the molar ratio of approx. 2 : 1, where the generation of the 90- and 30-kDa peptides reached the maximal value.

image

Figure 3. SDS/PAGE of α2M treated with cathepsin E or cathepsin D under acidic conditions.α2M from bovine (A) and human sources (B, C) was incubated with cathepsin E or cathepsin D at molar ration of 1 : 1 at 37 °C at the indicated pH values for various times, and then analyzed by SDS/PAGE under reducing conditions. A, bovine α2M at pH 3.8; B, human α2M at pH 3.8; C, human α2M at pH 4.5 and 5.5. A, 8% gel; B and C, 15% gel.

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image

Figure 4. Effects of substrate/enzyme ratios on generation of the cathepsin E-cleavage peptides of α2M. Bovine α2M was incubated with cathepsin E at various α2M/enzyme ratios at pH 3.8 and 37 °C for 30 min, and then the reaction products were analyzed by SDS/PAGE (6%) under reducing conditions.

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N-terminal sequencing of bovine α2M and sequencing its cDNA

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Purification of α2M from bovine serum
  6. Assays
  7. Sequencing of bovine α2M cDNA
  8. Gel electrophoresis and N-terminal sequence analysis
  9. Interaction of α2M with cathepsin E
  10. Analysis of synthetic peptides treated with cathepsin E and cathepsin D
  11. Results
  12. Effect of α2M on the hemoglobin hydrolyzing activity of cathepsin E and cathepsin D
  13. Effect of pH on the interaction of cathepsin E with α2M
  14. N-terminal sequencing of bovine α2M and sequencing its cDNA
  15. Analysis of the specific cleavage of α2M by cathepsin E with various synthetic peptides
  16. Structural changes in α2M upon complex formation with cathepsin E as determined by gel filtration analysis
  17. Discussion
  18. Acknowledgements
  19. References

Because the inhibition capacity of bovine α2M for cathepsin E was stronger than that of human α2M, and because there is no report on the sequence of the subunit of bovine α2M so far, we determined the partial amino-acid sequence of bovine α2M predicted from its cDNA sequence (corresponding to the residues 538–954 of human α2M), which contained the bait region. Comparison of the amino-acid sequence of bovine α2M determined with those of human, rat, and mouse origins revealed that the sequence was strongly related to those of these species (Fig. 5). Although the overall sequence of this region of bovine α2M was significantly similar to those of human, mouse, and rat sources (59%, 62%, and 55% identities, respectively), the bait region (corresponding to the residues 666–706 of human α2M) was dissimilar to those of other species and of different length. The N-terminal amino-acid sequences of the 90- (AVDGKEP) and 30-kDa peptides (LAIPVE) generated from cathepsin E-treated bovine α2M corresponded to the sequences of N-terminus and starting with 812th Leu of the intact α2M, respectively. It is worth emphasizing that the amino-acid sequences of cathepsin E-cleavage sites (shown by the box) were highly conserved among mammalian species. Taken together, these results indicate that cathepsin E specifically cleaved at the Phe811-Leu812 bond present in about 100mer downstream of the bait region of both bovine and human α2M.

image

Figure 5. Comparison of the partial amino-acid sequence of bovine α2M with those of human, rat, and mouse α2M species. The partial amino-acid sequence of bovine α2M, including the bait region to the thiol ester bond site, was shown aligned with those of human, rat and mouse α2M species. This region corresponds to the residues 538–954 of human α2M. Shading indicates identity relative to the bovine α2M sequence, and numbering is relative to the human α2M. The bait region is underlined and the thiol ester bond site is double-underlined. The arrow and the box indicate the cathepsin E cleavage site and the sequence used for peptide synthesis, respectively. Human, rat and mouse α2M sequences are from refs [45], [46] and [47], respectively.

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Analysis of the specific cleavage of α2M by cathepsin E with various synthetic peptides

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Purification of α2M from bovine serum
  6. Assays
  7. Sequencing of bovine α2M cDNA
  8. Gel electrophoresis and N-terminal sequence analysis
  9. Interaction of α2M with cathepsin E
  10. Analysis of synthetic peptides treated with cathepsin E and cathepsin D
  11. Results
  12. Effect of α2M on the hemoglobin hydrolyzing activity of cathepsin E and cathepsin D
  13. Effect of pH on the interaction of cathepsin E with α2M
  14. N-terminal sequencing of bovine α2M and sequencing its cDNA
  15. Analysis of the specific cleavage of α2M by cathepsin E with various synthetic peptides
  16. Structural changes in α2M upon complex formation with cathepsin E as determined by gel filtration analysis
  17. Discussion
  18. Acknowledgements
  19. References

To further confirm the specific cleavage of α2M by cathepsin E, synthetic peptides of α2M including the cathepsin E cleavage site region were designed and synthesized. These synthetic peptides were incubated with cathepsin E or cathepsin D at pH 3.8 and 37 °C for various time intervals and then the reaction products were analyzed by HPLC C18 column. The hexapeptide SPAFLA corresponding to the cleavage site of human α2M was efficiently cleaved by cathepsin E at the Phe-Leu bond, whereas it was not cleaved by cathepsin D (Table 1). The hepta peptide SSAFLAF corresponding to the cleavage site of bovine α2M was also cleaved by cathepsin E. However, differing from the peptide SPAFLA, this peptide was efficiently cleaved by cathepsin D. On the other hand, the pentapeptides SPAFL and SSAFL were not cleaved either cathepsin E or cathepsin D, indicating that the presence of Ala in the P′2 site is crucial for the selective cleavage of α2M by cathepsin E and that the addition of Phe to the P′3 site causes a loss of this selective action of cathepsin E. In agreement with these results, the cathepsin E-induced α2M degradation was significantly inhibited by these three peptides, most strongly by SPAFLA (Fig. 6).

Table 1. The ability of cathepsin E and cathepsin D to cleave synthetic peptides. The synthetic peptides (20 nm) containing the cathepsin E-cleavage site of α2M were incubated with or without either cathepsin E or cathepsin D (20 pm each) at pH 3.8 and 37 °C for the indicated time. The samples were then subjected to a HPLC C18 column chromatography and the resultant peak fractions were subjected to the amino-acid sequence analyzer. The values are expressed as a percentage of the initial intact peptide. The asterisk indicates the values for SPAF and SSAF generated by cleavage of SPAFLA and SSAFLAF, respectively. ND, not determined.
PeptidesCathepsin ECathepsin D
00.542400.524
SSAFL100NDND99100100100
SPAFL100NDND98100100100
SPAFLA10093632110010099
*SPAF073779001
SSAFLAF1002ND0100331
*SSAF099ND10006899
image

Figure 6. Effects of synthetic peptides on degradation of α2M by cathepsin E. Bovine α2M was incubated with cathepsin E at pH 3.8 and 37 °C for 30 min in the absence or presence of various synthetic peptides (500 µm), and then the reaction products were analyzed by SDS/PAGE under reducing conditions. Peptides used are Ser-Ser-Ala-Phe-Leu (1), Ser-Pro-Ala-Phe-Leu (2), and Ser-Pro-Ala-Phe-Leu-Ala (3).

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Structural changes in α2M upon complex formation with cathepsin E as determined by gel filtration analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Purification of α2M from bovine serum
  6. Assays
  7. Sequencing of bovine α2M cDNA
  8. Gel electrophoresis and N-terminal sequence analysis
  9. Interaction of α2M with cathepsin E
  10. Analysis of synthetic peptides treated with cathepsin E and cathepsin D
  11. Results
  12. Effect of α2M on the hemoglobin hydrolyzing activity of cathepsin E and cathepsin D
  13. Effect of pH on the interaction of cathepsin E with α2M
  14. N-terminal sequencing of bovine α2M and sequencing its cDNA
  15. Analysis of the specific cleavage of α2M by cathepsin E with various synthetic peptides
  16. Structural changes in α2M upon complex formation with cathepsin E as determined by gel filtration analysis
  17. Discussion
  18. Acknowledgements
  19. References

α2M was first incubated with cathepsin E (a molar ratio of 14 : 1) at pH values between 5.5 and 3.8 at various time intervals. The reaction mixtures were adjusted to pH 6.0 and then subjected to gel filtration on Superose 6. At every pH value, more than 80% of cathepsin E rapidly disappeared from the original position corresponding to a molecular mass as high as 80 kDa and appeared at the position where α2M was eluted. However, the α2M-cathepsin E complex was rapidly dissociated at pH 3.8 within a 2-min incubation (Fig. 7B). Parallel to this change, a significant amount of the original α2M disappeared and a few protein peaks were produced. After the 1-h incubation at pH 3.8, the original α2M completely disappeared and generated two major protein peaks at the positions corresponding to molecular masses as high as 90 and 30 kDa. In agreement with this change cathepsin E reappeared at the original position with no loss of activity. On the other hand, the dissociation of cathepsin E from the α2M complex was relatively slow at pH 4.5 and 5.5 (Fig. 7A). Although the formation of α2M–cathepsin E complex was rapid at pH 5.5 and 4.5, the rate of dissociation of this complex was pH-dependent. On the other hand, the antiproteolytic activity of α2M for trypsin was lost by incubation with cathepsin E in a dose-dependent manner (Fig. 8).

image

Figure 7. Gel filtration on Superose 6 of α2M treated with cathepsin E at various pH values. Bovine α2M was incubated with cathepsin E at 37 °C at the indicated pH values. The reaction mixtures were adjusted to pH 6.0 and then run on a Superose 6 column equilibrated with 10 mm sodium phosphate buffer, pH 6.0, containing 150 mm NaCl. Fractions were analyzed by the cathepsin E activity at pH 3.8 with the synthetic substrate MOCAc-Gly-Lys-Pro-Ile-Ile-Phe-Phe-Arg-Leu-Lys(Dnp)-d-Arg-NH2, SDS/PAGE and immunoblotting with antibodies to cathepsin E. (a) The elution profiles of α2M and cathepsin E when each protein was run on the column independently. The α2M was treated with cathepsin E at pH 4.5 for 1 h (b), at pH 5.5 for 1 h (c), at pH 3.8 for 45 s (d), at pH 3.8 for 2 min (e), and at pH 3.8 for 1 h (f). The solid and dotted lines illustrate the cathepsin E activity and the absorbance at 280 nm, respectively.

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image

Figure 8. Effect of cathepsin E-treated α2M on the antiproteolytic activity for trypsin. Bovine α2M was incubated with cathepsin E at various enzyme/α2M ratios at pH 3.8 and 37 °C for 30 min, and then the reaction products were neutralized and analyzed for the antiproteinase activity on trypsin using 1% casein as a substrate. The values are expressed as percentages of the trypsin activity in the absence of α2M.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Purification of α2M from bovine serum
  6. Assays
  7. Sequencing of bovine α2M cDNA
  8. Gel electrophoresis and N-terminal sequence analysis
  9. Interaction of α2M with cathepsin E
  10. Analysis of synthetic peptides treated with cathepsin E and cathepsin D
  11. Results
  12. Effect of α2M on the hemoglobin hydrolyzing activity of cathepsin E and cathepsin D
  13. Effect of pH on the interaction of cathepsin E with α2M
  14. N-terminal sequencing of bovine α2M and sequencing its cDNA
  15. Analysis of the specific cleavage of α2M by cathepsin E with various synthetic peptides
  16. Structural changes in α2M upon complex formation with cathepsin E as determined by gel filtration analysis
  17. Discussion
  18. Acknowledgements
  19. References

α2M has a wide range of physiological activities via its interaction with various target proteins, such as the control of the activity of proteinases, the regulation of the activities of numerous cytokines and growth factors, and the enhancement of antigen presentation. Once taken up inside the cell the α2M–protein complexes are rapidly degraded in the endolysosome system. However, the fate of α2M in the complexes and the nature of its degradation are not clear. The present findings on the biochemical nature of the interaction of cathepsin E with α2M are unique and unexpected. This is the first report of the structural and functional disruption of α2M integrity by cathepsin E. It is surprising that cathepsin E can be associated with α2M at very low pH values and rapidly cleaves it at the specific site distinct from the bait region. Under mild acidic conditions, α2M appears to interact with cathepsin E [35], as well as other proteinases, to be cleaved a peptide bond in the bait region and thereby undergoes a conformational change similar to that occurring at neutral pH values [42], where cathepsin E is essentially inactive [27,36]. However, at pH values below 5.0, α2M is unstable and is likely to lose the proteinase-binding activity [43], where cathepsin E is more active. The endolysosomal compartment is the major site of endogenous protein degradation. Degradation of α2M, like other endocytosed proteins, is known to occur rapidly inside the endolysosomes, where the pH is maintained below 5.0. The intravacuolar pH is important because most of the endolysosomal hydrolases including cathepsins E and D require a pH of 3.5–5.0 for maximal activity. Similarly, microbicidal systems such as peroxidase-hydrogen peroxide need such an acid pH for optimal activity [44].

At pH values between 3.8 and 5.5, cathepsin E selectively bound α2M and cleaved it at the Phe811-Leu812 bond at a distance from the bait region. Therefore, the cathepsin E–α2M interaction below pH 5.0 appears to be unique and is different from that occurring at mild acidic and neutral pH values. Namely, cathepsin E is efficiently associated with α2M at pH values below 5.0 without loss of the proteolytic activity and rapidly cleaves it at the Phe811-Leu812 bond distinct from the bait region and then leaves the associated site. Considering that a very low pH treatment results in dissociation of α2M into the dimers, which do not reassociate normally but tend to aggregate [43], it is more likely that cathepsin E interact with the distinct region from the bait region of the α2M. Under these conditions, however, the analogous aspartic proteinase cathepsin D neither interacts with α2M nor cleaves it. As previous work has demonstrated that the action of cathepsin D, as well as cathepsin E, toward protein substrates was blocked by α2M at pH 6.2 [34], a slight decrease in pH below 5.5 is very likely to cause an additional conformational change of α2M and thereby abolish the ability of cathepsin D to bind α2M.

This study also described for the first time the cloning and sequencing of partial cDNA for bovine α2M, as its primary structure is likely to provide significant information regarding the finding that bovine α2M is more sensitive to cathepsin E digestion than that from other species (data not shown). Analysis of the amino-acid sequence of bovine α2M deduced from isolated cDNA clones revealed that its gross structure was homologous to those of human, rat, and mouse α2M, although the bait region was dissimilar to that of either α2M from other species. In particular, the sequence around the cathepsin E cleavage site (Phe811–Leu812 bond) was highly conserved in all of the other species. To further confirm the selective cleavage of the Phe811–Leu812 bond in α2M by cathepsin E, we synthesized some peptides corresponding to part of the cleavage site and analyzed the susceptibility of these peptides to cleavage by cathepsin E. The hexapeptide SPAFLA corresponding to part of the cathepsin E cleavage site (the Ser808–Ala813 sequence of human α2M) was selectively cleaved at the Phe-Leu bond by cathepsin E, but not cathepsin D. The peptides SPAFL and SSAFL corresponding to the sequence Ser808–Leu812 of human and bovine α2M, respectively, were not cleaved by either cathepsin E or cathepsin D. In agreement with the results, the peptide SPAFLA significantly inhibited the degradation of α2M by cathepsin E. These results indicate that the presence of Ala in the P′2 is essential for selective cleavage the synthetic peptides by cathepsin E. The presence of Pro in the P3 site in α2M, however, is unlikely to be crucial for its selective cleavage, as the 809th residue in bovine α2M is Ser in place of Pro found in other species.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Purification of α2M from bovine serum
  6. Assays
  7. Sequencing of bovine α2M cDNA
  8. Gel electrophoresis and N-terminal sequence analysis
  9. Interaction of α2M with cathepsin E
  10. Analysis of synthetic peptides treated with cathepsin E and cathepsin D
  11. Results
  12. Effect of α2M on the hemoglobin hydrolyzing activity of cathepsin E and cathepsin D
  13. Effect of pH on the interaction of cathepsin E with α2M
  14. N-terminal sequencing of bovine α2M and sequencing its cDNA
  15. Analysis of the specific cleavage of α2M by cathepsin E with various synthetic peptides
  16. Structural changes in α2M upon complex formation with cathepsin E as determined by gel filtration analysis
  17. Discussion
  18. Acknowledgements
  19. References
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